The Trypanosoma cruzi Proteome

J. A. Atwood III et al.

Science 309, 473 (2005);
DOI: 10.1126/science.1110289

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The Trypanosoma cruzi Proteome

J. A. Atwood III,1* D. B. Weatherly,2,3* T. A. Minning,2,3 B. Bundy,2,3
C. Cavola,1 F. R. Opperdoes,4 R. Orlando,1 R. L. Tarleton2,3.
To complement the sequencing of the three kinetoplastid genomes reported
in this issue, we have undertaken a whole-organism, proteomic analysis of the
four life-cycle stages of Trypanosoma cruzi. Peptides mapping to 2784 proteins
in 1168 protein groups from the annotated T. cruzi genome were identified
across the four life-cycle stages. Protein products were identified from 91000
genes annotated as hypothetical in the sequenced genome, including members of a newly defined gene family annotated as mucin-associated surface
proteins. The four parasite stages appear to use distinct energy sources, including histidine for stages present in the insect vectors and fatty acids by intracellular amastigotes.
Trypanosoma cruzi exists in four morphologically and biologically distinct forms during
its cycle of development in mammals and
insects (Fig. 1). Metacyclic trypomastigotes
develop in the hind gut of triatomine insect
vectors and initiate infection in a wide variety of animal species, including humans. In
host cells, trypomastigotes convert to replicative amastigote forms that reside in the
host-cell cytoplasm. After multiple rounds of
binary fission, the aflagellate amastigotes convert into flagellated trypomastigotes that burst
from the host cell and circulate in the bloodstream. There, the trypomastigotes can invade
other host cells and thus spread the infection
throughout the body. Alternatively, trypomastigotes acquired during a blood meal convert
to epimastigote forms, which replicate in the
insect gut before eventually differentiating
into infective metacyclic trypomastigote forms.
Drugs for the treatment of T. cruzi infection are
inadequate, and vaccines are lacking. Like
other trypanosomatids, T. cruzi appears to regulate protein expression primarily posttranscriptionally through variations in mRNA stability
or the translational efficiency of mRNAs (1).
This limits the use of DNA microarrays (25)
and makes proteomic analysis especially attractive for examining global changes in protein
expression during development in T. cruzi.
Metacyclic trypomastigotes, amastigotes, trypomastigotes, and epimastigotes of T. cruzi were
isolated, and proteins were extracted from
whole-cell or subcellular lysates (fig. S1)
(6). Peptides generated by digestion of the
whole-cell or subcellular lysates were independently separated and analyzed at least in
Complex Carbohydrate Research Center, 2Center for
Tropical and Emerging Global Diseases, 3Department
of Cellular Biology, University of Georgia, Athens, GA
30602, USA. 4Research Unit for Tropical Diseases and
Laboratory of Biochemistry, Christian de Duve Institute of Cellular Pathology, and Catholic University of
Louvain, Brussels, Belgium.

duplicate by offline multidimensional liquid

chromatography, online reverse-phase liquid
chromatography and tandem mass spectrometry
(LC-MS/MS) (6, 7). A total of 602 tryptic
peptide samples were analyzed, generating
139,147 tandem mass spectra. Because of
differences in protein recovery from the four
life-cycle stages, trypomastigote and amastigote
stages are undersampled relative to metacyclic
trypomastigotes and epimastigotes (table S1). A
total of 5,720 unique peptides were matched
with high confidence to 1168 protein groups
containing 2784 total proteins using the Mascot
search engine and PROVALT parsing and
clustering tools (7), as described in (6) (table
S2). The approach of grouping protein isoforms
(8, 9) is particularly important in T. cruzi
because the genome contains multiple, non-

identical copies of many genes, including a

number of large gene families with hundreds of
distinct members (10). In addition, the T. cruzi
CL Brener strain used for the sequencing
project is a hybrid of two genotypes and thus
has multiple distinct alleles for most genes.
Table 1 summarizes the proteins assigned to
each life-cycle stage. Nearly 30% (838 of 2784)
of the identified proteins, including most of the
proteins previously documented or expected to
be produced in the greatest abundance, were
detected in all life-cycle stages.
Although there are limitations in the ability of shotgun proteome LC-MS/MS analysis
to detect precise changes in protein levels, it
is possible to track the relative abundance of
proteins in the four T. cruzi stages using measures of protein coverage (11). Among the topscoring proteins in all four T. cruzi proteomes
are many housekeeping proteins that are also
among the highest ranking proteins in yeast
(12). However, many other highly abundant
proteins in the T. cruzi proteome are either
absent in the yeast genome or are expressed
at very different relative levels in these two
eukaryotes (see specific examples in supporting online text). Table 2 summarizes some of
the major protein groups and families identified in the T. cruzi proteome. These data
reflect a combination of the relative abundance of the proteins comprising each group,
the size of gene families, and the ease with
which certain proteins can be detected by
LC-MS/MS analysis (supporting online text).
Of the 2784 total proteins identified in this
analysis, 1008 are from genes annotated as

*These authors contributed equally to this work.

.To whom correspondence should be addressed.
E-mail: tarleton@cb.uga.edu

Fig. 1. Life cycle and summary of the major findings of proteome analysis in T. cruzi. T. cruzi
trypomastigotes circulate in the blood of infected hosts, including humans, but must enter host cells
(oftentimes muscle cells) and convert to amastigote forms to replicate. Triatomine bug vectors
become infected by ingesting trypomastigotes during the course of a blood meal on infected
mammalian hosts. Conversion of the trypomastigotes into epimastigotes, replication of these
epimastigotes, and their eventual transformation into metacyclic trypomastigotes, occurs in the
insect gut. Metacyclic trypomastigotes initiate new infection in mammals when infected insects
are ingested or by deposition of parasites in the feces, usually during a blood meal.

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hypothetical,[ validating these as bona fide
genes in T. cruzi. More than half of these hypothetical genes have orthologs in the Leishmania major and/or T. brucei genomes.
T. cruzi trypomastigotes circulate in the
blood, where they are exposed to host immune
effector molecules, including specific antibodies.
Unlike the related African trypanosomes, T.
cruzi trypomastigotes do not undergo antigenic
variation but instead express on their surface
multiple members of several large families of
molecules; the best characterized of these are
the mucin and trans-sialidase (ts) families (13).
Thirty of the 50 top-scoring proteins detected
exclusively in trypomastigotes are ts family
members. Likewise, the amastigote and metacyclic stages appear to express subsets of ts
molecules unique to each stage, whereas no ts
expression was detected in the epimastigote
proteome (Fig. 2 and table S2). Trans-sialidase
enzymatic activity is reportedly present in only
a small subset of the 91000 ts proteins encoded
in the T. cruzi genome, and it has been linked to
the presence of Tyr342 in the catalytic Nterminal region and SAPA repeats in the C
terminus (13). Among the 223 ts proteins
detected in the proteome are the products of
all 15 genes predicted to encode enzyme-active
ts. The production of a large number of nonenzymatic ts family members coincident with
these ts enzymes may deflect immune responses
away from the enzymatically active targets or
may provide a pool of altered peptides that
could antagonize T cell responses (14).
In addition to the ts and mucin families,
the T. cruzi genome contains several other
high-copy multigene families (Table 2 and
supporting online text). We detected expression of several mucin-associated surface proteins (MASPs), a gene family first discovered
as part of the sequencing and annotation effort
(10), predominantly in the trypomastigote
proteome. Like proteins from the other multigene families in T. cruzi, many MASP family
members have predicted signal sequences
and glycosylphosphatidylinositol anchor addition sites and thus are likely to be surface
expressed. Nine MASP gene family proteins
were identified in our analysis, each by only a
single peptide match. This result suggests either
that MASPs are not as abundantly expressed as
the trans-sialidase proteins or that, like the
mucins, MASPs have extensive posttranslational modifications that complicate their detection by shotgun proteomics. However, detection
of the MASPs in the relatively undersampled
trypomastigote stage suggests that they are not
minor constituents of the T. cruzi proteome.
The transition from trypomastigote to
amastigote can be stimulated extracellularly
by simulating the low pH environment of the
phagosomal/lysosomal compartment that T.
cruzi initially encounters upon cell entry (15),
making early time points in the transformation
process to the amastigote stage amenable to

474

transcriptome and proteome analysis. The results from this proteome analysis of amastigotes are in agreement (with one exception)
with the restricted data set generated by comparison of trypomastigotes and early-stage
amastigotes using DNA microarray analysis
(3) (table S4), further supporting the quality of
this analysis. In addition to the expression of a
distinct subset of trans-sialidasefamily genes,
many of which are related to the amastigote
surface protein 2 molecule previously reported
to be preferentially expressed in amastigotes
(16) (Fig. 2), the transition of trypomastigotes
to amastigotes also appears to be accompanied
by a dramatic shift from carbohydrate- to lipiddependent energy metabolism (table S3). This
is demonstrated by the virtual absence of glucose transporters and the detection of enzymes
that oxidize fatty acids to give acetylcoenzyme
A. Enzymes of the citric acid cycle, which
oxidize acetyl coenzyme A to carbon dioxide
and water, are also abundant in amastigotes.
Amastigotes are likely to be dependent on gluconeogenesis for the synthesis of glycoproteins and glycoinositolphospholipids (GIPLs),

and aspartate aminotransferases (4698.t00001,

4779.t00007) specific to amastigotes may be
important in this process. These proteins lack
the mitochondrial targeting signal present on
the aspartate aminotransferase expressed in all
stages (6015.t00007) and thus likely reside in
the cytoplasm. Mitochondrially produced oxaloacetate, after transamination, may be transported to the cytosol by a malate/aspartate
shuttle and then converted by the cytosolic
aspartate aminotransferase and a phosphoenol
pyruvate carboxykinase into phosphoenol pyruvate, the substrate for gluconeogenesis.
In addition to several heat-shock proteins and
kinases, among the other proteins detected
preferentially or exclusively in amastigotes
are a group involved in endoplasmic reticulum
(ER) to Golgi trafficking, including rab1
(4703.t00005), sec23 (8726.t00010), and sec31
(6890.t00029). The detection of this set of
proteins involved in vesicular trafficking in
amastigotes but not in the more highly sampled
metacyclic and epimastigote stages suggests
a more active trafficking process or the preferential use of selected rab and sec proteins in

Table 1. Protein group and protein identifications for each developmental stage.
Protein groups
(proteins)
29
21
44
335
27
65
146
24
43
47
53
12
187
92
43
1168

(49)
(41)
(161)
(838)
(84)
(110)
(538)
(50)
(125)
(122)
(93)
(22)
(315)
(162)
(74)
(2784)

Amastigote
X
X
X
X
X
X
X
X

Trypomastigote

Metacyclic
trypomastigote

X
X
X
X

X
X

Epimastigote

X
X

X
X
X
X
X
X
X

691 (1871)

582 (1486)

X
X

X
X

X
X

X
X

X
X
732 (1861)

969 (2339)

Table 2. Major protein families and functional classes.

Number of identified proteins
Protein functional classes
Ribosomal
Proteasome/Ubiquitin
Heat shock/Chaperonins
Translation/Transcription
Histones
Gene families
Trans-sialidase
RHS
GP63
Cysteine protease
MASP
Mucins
Hypothetical genes
Hypothetical
Hypothetical conserved
Hypothetical to be annotated

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212
67
61
49
36
223
399
29
30
9
0
155
505
348

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REPORTS
amastigotes (table S3). We also extend the data
on the selective expression in amastigotes and
epimastigotes of several ABC transporters
(7164.t00003, 8319.t00008) that are hypothesized to have a role in cargo selection and/or
vesicular transport in trypanosomes (17). A putative lectin (6865.t00003) with homology to
ERGIC (ER Golgi intermediate compartment),
a protein involved in cargo selection in coat
protein complex II vesicles, is also detected in
trypomastigotes and amastigotes but not in
metacyclic or epimastigote forms.
In contrast to both T. brucei and L. major,
the T. cruzi genome encodes enzymes capable
of catalyzing the conversion of histidine to glutamate. The first two enzymes in this pathway,
histidine ammonia-lyase (6869.t00022) and
urocanate hydratase (4881.t00011), are abundant in the insect stages but nearly undetectable
in the mammalian stages (only a single
spectrum matching histidine ammonia-lyase in
amastigotes), consistent with the functioning of
this pathway primarily in epimastigotes and
metacyclic trypomastigotes. This expression
pattern is notable, given that histidine is the
dominant free amino acid in both the excreta
and the hemolymph of Rhodnius prolixus
(18, 19), a well-studied vector for T. cruzi. The
abundance of histidine in this and other bloodfeeding insects likely reflects the high histidine
content of hemoglobin (20). Thus, T. cruzi
epimastigotes seem particularly adapted among
the kinetoplastids to take advantage of this
plentiful energy source in the gut of its insect
vector. This is analogous to the use of proline
as an energy source by T. brucei (21).
The transformation of epimastigotes to
metacyclic trypomastigotes is accompanied
by the production of a number of key en-

zymes and substrates important in antioxidant

defense in T. cruzi. The H2O2 and peroxynitrite detoxifying enzymes ascorbate peroxidase (6846.t00006, 4731.t00003) (22) and the
mitochondria-localized tryparedoxin peroxidase (8115.t00003) are both elevated after
epimastigote to metacyclic conversion, as
are tryparedoxin (5824.t00003), the substrate
for tryparedoxin peroxidase, and the enzymes trypanothione synthase (8070.t00009,
7998.t00005) and iron superoxide dismutase
(5781.t00004), responsible for synthesis of
trypanothione and for the conversion of superoxide anion to hydrogen peroxides, respectively. These changes are consistent with a
preadaptation of metacyclic forms to withstand the potential respiratory burst of phagocytic cells in the mammalian host. Enzymes of
the pentose-phosphate shunt aid this process
through the production of the nicotinamide
adenine dinucleotide phosphate required for
the reduction of trypanothione. Also noticeable in the transition of epimastigotes into
metacyclic trypomastigotes is a substantial
decrease in the representation of ribosomal
proteins in the metacyclic proteome; 37 of the
50 highest scoring proteins in the epimastigote
proteome that are not detected in the metacyclic
trypomastigote proteome are ribosomal proteins. A reduction in the capacity for protein
production would be consistent with the stationary, nonreplicating status of metacyclic trypomastigotes. DNA microarray analysis has also
documented a substantial down-regulation of
ribosomal protein expression in metacyclic
forms in L. major (23).
A search for peptides with modifications
(e.g., acetylations, methylations, or phosphorylations) resulted in 8 additional protein iden-

Fig. 2. Stage-specific detection

of ts proteins. Cumulative protein
scores based on summing the Mascot scores for all high-confidence
peptides are used to display the
stage-regulated expression of ts
proteins detected in the proteomes.
Peptides matching to 223 members of the ts family clustered
into 47 protein groups; only the
top-scoring protein for each protein group is shown. Most ts proteins are detected exclusively in
one stage. A, amastigote; T, trypomastigote; M, metacyclic trypomastigote; E, epimastigote.

tifications and the detection of modifications on

81 previously identified proteins (supporting
online text and table S5). To identify additional
genes potentially missed in the annotations
provided by the T. cruzi sequencing consortium, a database of approximately 817,000 open
reading frames (ORFs) of 950 amino acids
was constructed and screened using spectra that
failed to match proteins predicted by the annotated genome (6). This analysis yielded 79
new genes, new alleles, or modifications to existing gene annotations (table S6). Sixty-six of
these ORFans are new alleles of annotated genes
or corrections to existing annotations, which
suggests that the prediction models and annotations of The Institute for Genomic Research
Seattle Biomedical Research InstituteKarolinska
Institutet Trypanosoma cruzi Sequencing Consortium (TSK-TSC) have been extremely efficient in accurately predicting genes. In all cases,
these new annotations map to the Bcoding[
strand of DNA among genes that are part of
polycistronic units. This result is consistent with
the model of kinetoplastid genes being clustered
in large transcriptional units on the coding
strand of DNA (24). Strand-switch regions separate these clusters and allow for changing of
the coding strand at sites of transcription initiation. Thus, although transcriptional activity
on the Bnoncoding[ DNA strand has been documented (25), the proteome does not provide
evidence for translation of those alternative
strand transcripts.
High-throughput proteome analyses are inherently incomplete, because the available
methodologies do not have sufficient dynamic
range to identify and quantify all proteins
expressed in an organism. In this analysis, nearly
50% of all the spectra matching to proteins
mapped to the 67 most abundant protein groups.
A higher number of lower abundance proteins
can likely be revealed by depleting these highly
abundant proteins before whole proteome analysis. Analysis of the proteomes of T. cruzi
reveals the operation of several previously undocumented stage-specific pathways that could
be appropriate targets for drug intervention.
Among the most interesting of these are the
proposed pathways for energy generation in
amastigotes and epimastigotes. Additionally, the
identification of the proteins expressed in abundance in trypomastigotes and amastigotes of T.
cruzi provides a substantial new resource of candidates for vaccine development. This proteome
analysis of T. cruzi also validates the high quality
of the gene predictions generated by the TSKTSC by confirming the expression of 91000
hypothetical genes and at the same time revealing G15 genes missed in the initial annotation.
References and Notes
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Tarleton, Mol. Biochem. Parasitol. 131, 55 (2003).

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Anal. Chem. 75, 4646 (2003).
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Nussenzweig, Parasitology 110, 547 (1995).
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The authors thank the TSK-TSC for providing access
to genome sequence data before and after assembly
and public release of an annotated genome, K. Tyler
and D. Hoft for assistance in the production of metacyclic stages, L. Cheng for technical assistance, C.
Reese for artwork, B. Striepen for comments on
the manuscript, and S. Kring Sullivan for early
work on mass spectrometric analysis of T. cruzi. This
work is supported by NIH grant PO1 AI044979 to
R.L.T. Data on all peptides mapping to annotated
genes is available on TcruziDB (http://tcruzidb.org).

Tau Suppression in a
Neurodegenerative Mouse
Model Improves Memory Function
K. SantaCruz,1* J. Lewis,5* T. Spires,6* J. Paulson,2 L. Kotilinek,2
M. Ingelsson,6 A. Guimaraes,2 M. DeTure,5 M. Ramsden,2
E. McGowan,5 C. Forster,1 M. Yue,5 J. Orne,6 C. Janus,5 A. Mariash,2
M. Kuskowski,7 B. Hyman,6 M. Hutton,5 K. H. Ashe2,3,4,7.
Neurofibrillary tangles (NFTs) are the most common intraneuronal inclusion in
the brains of patients with neurodegenerative diseases and have been
implicated in mediating neuronal death and cognitive deficits. Here, we found
that mice expressing a repressible human tau variant developed progressive
age-related NFTs, neuronal loss, and behavioral impairments. After the
suppression of transgenic tau, memory function recovered, and neuron numbers
stabilized, but to our surprise, NFTs continued to accumulate. Thus, NFTs are
not sufficient to cause cognitive decline or neuronal death in this model of
tauopathy.
Neurofibrillary tangles are composed of filaments of hyperphosphorylated tau (14), a
microtubule-associated protein (5). They correlate well with cognitive deficits (68) and
neuron loss (9), and they have been implicated
in mediating neurodegeneration and dementia
in Alzheimer_s disease (AD) (1013) and other
tauopathies (14). Transgenic mice expressing
human tau have consistently demonstrated
neurological deficits and neuron loss appearing
with NFTs (1519). The association between
NFTs, neuron loss, and brain dysfunction in
1

Department of Laboratory Medicine and Pathology,

Department of Neurology, 3Department of Neuroscience, and 4Graduate Program in Neuroscience,
University of Minnesota Medical School, Minneapolis,
MN 55455, USA. 5Department of Neuroscience,
Mayo Clinic Jacksonville, Jacksonville, FL 32224,
USA. 6Department of Neurology, Massachusetts
General Hospital, Charlestown, MA 02129, USA.
7
Geriatric Research, Education, and Clinical Center
(GRECC), Minneapolis VA Hospital, Minneapolis, MN
55417, USA.
2

*These authors contributed equally to this work.

.To whom correspondence should be addressed.
E-mail: hsiao005@umn.edu

476

humans and mice has led to the belief that

NFTs invariably cause brain dysfunction and
neurodegeneration. However, this idea has
never been rigorously tested. We hypothesized
that examining the effects of suppressing the
tau transgene would elucidate the dependence
of functional changes on the structural and
biochemical abnormalities related to abnormal
tau expression for two reasons: It would
provide an opportunity to evaluate the dependence of specific structural and functional abnormalities on the continuous expression of the
tau transgene, and it would distinguish reversible from irreversible lesions.
We created transgenic mice expressing
mutant tau that could be suppressed with
doxycycline (fig. S1A). A responder transgene
was generated consisting of a tetracyclineoperonresponsive element (TRE) placed upstream of a cDNA encoding human four-repeat
tau with the P301L mutation (4R0N tauP301L)
that is linked to hereditary tauopathy (20). An
activator transgene in a second mouse line
consisted of the tet-off open reading frame (21)
placed downstream of Ca2calmodulin ki-

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Raw data in either the original peak-list (.PKL) format or

in mzData XML format (Minimum Information About a
Proteomics Experiment standard) can be downloaded
from http://kiwi.rcr.uga.edu/tcprot/downloads.html.
Complete lists of all peptides identified, prerun queries
identifying proteins expressed in specific life-cycle stages,
and tools to query and view these data are also available
on http://tcruzidb.org and/or http://kiwi.rcr.uga.edu/
tcprot.
Supporting Online Material
www.sciencemag.org/cgi/content/full/309/5733/473/
DC1
Materials and Methods
SOM Text
Figs. S1 and S2
Tables S1 to S6
References
26 January 2005; accepted 13 June 2005
10.1126/science.1110289

nase II promoter elements, which resulted in

expression from the TRE that was restricted to
forebrain structures (22). Mice harboring responder or activator transgenes were bred to
generate bigenic progeny containing both transgenes. As expected, transgenic tau mRNA expression in bigenic mice was largely restricted
to structures in the forebrain (Fig. 1A).
To ascertain the relation between tauP301L
dosage and the development of neurofibrillary
pathology, we examined two lines of mice
expressing 7 and 13 units of tauP301L (one
unit is equivalent to the level of endogenous
murine tau) (Fig. 1B). The rate at which mice
developed neurofibrillary pathology was directly related to the amount of tauP301L expression. Mice with 7 units of tauP301L
showed accumulation of hyperphosphorylated tau in cortical neurons (pretangles) by
14.5 months of age, but did not develop argyrophilic tangle-like inclusions until 20 months
of age. Mice with 13 units of tauP301L,
rTg(tauP301L)4510 (r for regulatable), developed pretangles, which we detected using immunohistochemistry with multiple antibodies
to human phospho-tau epitopes, as early as
2.5 months of age. Pathology progressed rapidly in these mice (Fig. 1C). Argyrophilic
tanglelike inclusions appeared in the cortex by
4 months and in the hippocampal formation by
5.5 months. The neuronal inclusions in this
line were composed of a mass of straight tau
filaments and are henceforward referred to as
NFTs (Fig. 1D). A significant loss in brain
weight was evident by 5.5 months (Fig. 2A),
as well as significant decreases (60%) in
total numbers of CA1 hippocampal neurons
(Fig. 2B). The loss in brain weight and neurons
progressed in 7- and 8.5-month-old mice, with
23% of CA1 pyramidal cells remaining at
8.5 months. Gross atrophy of the forebrain was
evident in a 10-month-old mouse (Fig. 1E).
Thus, the expression of tauP301L was sufficient
to produce an age-related loss of neurons and
generalized forebrain atrophy, with concomitant abnormal accumulation of hyperphosphorylated tau lesions.

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